All of the structural and regulatory genes of simian immunodeficiency virus isolated from Macaca nemestrina (SIVmne) were cloned into mammalian cell expression vectors (plasmids) and constructs were optimized for maximal expression in vitro and immunogenicity in mice. Four M. fascicularis were immunized intradermally and intramuscularly four times over 36 weeks with plasmid DNA, and four received two DNA priming inoculations followed by two boosts of recombinant gp160 plus Gag-Pol particles in MF-59 adjuvant. Four controls received vector-only DNA or adjuvant-only immunizations. Following intrarectal challenge with SIVmne, all macaques became infected. Three of four monkeys immunized with DNA alone maintained low plasma virus loads by one year post-challenge; only one animal exhibited significant CD4+ cell decline. Two of the DNA-plus-boost and three control macaques had high virus loads and associated CD4+ cell decline. The total virus load for all macaques in the DNA-vaccin ated group for the first year post-challenge was significantly lower than that in the control group. Humoral immune responses were elicited in all vaccinees, but were higher in animals receiving a protein boost. Both vaccine protocols elicited comparable helper T cell (Th) proliferative responses to gp160. Measurement of cytokine message in activated peripheral blood mononuclear cells (PBMC) at time of challenge suggested a dominant Th1 state in three of four DNA-immunized animals and one of the DNA-plus-boost animals; this status correlated with low virus loads and high CD4+ cell counts post-challenge. Combination immunization with DNA-based multigenic vaccines and multiple antigen protein boosting offers a promising alternative to live-attenuated virus vaccination for protection against AIDS in the simian model. FUNDING RR00166 and AI26503. Haigwood, N.L. and Zolla-Pazner, S. Humoral immunity to HIV, SIV, and SHIV. AIDS 12 S121-S132, 1998. Mossman, S.P., Pierce, C.C., Robertson, M.N., Watson, A.J., Montefiore, D.C., Rabin, M., Lynch, J.B., Kuller, L., Thompson, J., Morton, W.R., Benveniste, R.E., Hu, S-L., Greenberg, P., and Haigwood, N.L. Immunization against SIVmne in macaques using multigenic DNA vaccines. Proc.16th Ann. Symp. Nonhuman Primate Models for AIDS, Abstract #35, 1998.